CZ282772B6 - Process of fermentation industrial production of phytase enzyme preparation - Google Patents

Abstract

This method of producing a phytase enzyme preparation by industrial fermentation through the submerged cultivation of an Aspergillus strain on a medium containing starch with a growth limitation with phosphorus, consisting of the submerged cultivation of the strain of the Aspergillius niger 921 CCF 2951 at a temperature of 25-27 degrees C, at a pH of 2.0 to 6.0, particularly 2.5 to 3.5, with aeration and agitation providing an oxygen concentration of 10-99% of the coefficient of the dissolubility of the gases, mainly of 20-60%, with the subsequent separation of the fungal mycelium, dialysis and ultrafiltration of the supernatant and lyophilisation of the concentrated solution of enzymes.

Description

Technical field

BACKGROUND OF THE INVENTION The present invention relates to a process for the fermentative industrial production of an enzyme preparation of phytase (myoinositol hexakisphosphate phosphohydrolase), which is useful in animal production for reducing phosphorus in animal excrements, reducing indigestible phytin in food, purified phytases, acid phosphatases and phosphorylated compounds.

BACKGROUND OF THE INVENTION

Phytase is used in animal production in monogastric animals to cleave phytin contained in compound feed, reduce phosphorus content in animal excrements and thereby prevent the eutrophication of waters in areas with developed livestock production, improve digestive enzyme activity and optimize utilization of vital ions, especially calcium (phytin is a strong chelating agent).

The phytase is produced by fermentation using some species of Aspergillus fungi by GistBrocades, NL, Alltech, USA and Alco Ltd. Biotechnology, Finland. In order to achieve the highest productivity of the production system, it is desirable to work with both the high-production strain of the microorganism and the fermentation and isolation technology ensuring a high yield of the active enzyme. However, available scientific literature suggests that phytase activities achieved using the known Aspergillus ficuum production strains (NRRL 3135 18-20 nkat / mL, Ullah and Gibson 1987 or genetically bred strain 100-127 nkat / mL, Hartingsveldt et al. 1993) are lower than the activity achieved by our production strain Aspergillus niger 921, CCF 2951, ie 150-160 nkat / mL. The process of the prior art production of phytase with other Aspergillus strains is thus more economically demanding.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a process for the fermentative industrial production of a phytase enzyme preparation by submerged Aspergillus strain on starch-containing medium while limiting phosphorus growth according to the invention in that the Aspergillus niger 921 CCF 2951 strain is sub-grown at 2.0-6.0 (especially at 2.5-3.5) with aeration and stirring providing an oxygen concentration of 10-99% gas solubility coefficient, especially at 20-60%, followed by mycelium separation of the fungus, dialysis and ultrafiltration supernatant and lyophilizing the concentrated enzyme solution by ultrafiltration. In addition to the preponderance of phytase, the enzyme preparation also contains non-specific acid phosphatases and α-amylase.

The nitrogen source in the culture medium is 0.5-1 wt. sodium nitrate, ammonium sulfate or ammonium chloride. During cultivation of Aspergillus niger 921, CCF 2951 strain on 0.5-1% sodium nitrate, sulphate or ammonium chloride medium, the pH of the medium is maintained at 2.5-3.5, preferably a constant value of 3.0, by automatic dosing of potassium, sodium or ammonium hydroxide solutions.

The corn starch content in the medium is in the range of 2-8% by weight, preferably for the preparation of inoculum 5-9% by weight. and for self-cultivation in production fermenters 4-8 wt.

The high-production strain Aspergillus niger 921, CCF 2951 is cultivated in production bioreactors, in addition to a one-time process, semi-continuously under semi-sterile to non-sterile conditions.

Separation of fungal mycelium at the end of cultivation can also be carried out in two stages, sedimentation and separation by centrifugation or filtration.

The supernatant after mycelial separation is dialyzed to a residual carbohydrate value of 0-3 g / L, preferably to 2.0 g / L.

The dialysis and ultrafiltration of the fermented medium is carried out on various types of devices and membranes providing retention of protein molecules of 10-100 kDa, preferably 50 kDa.

A dry enzyme preparation containing in addition to phytase also non-specific acid phosphatase and α-amylase is adapted for nutritional purposes to the desired phytase activity.

The technological process for the production of the mixed phytase preparation was obtained by optimizing the conditions of all stages of production, ie strain cultivation, separation, dialysis, ultrafiltration and lyophilization.

The advantages of the process of producing the phytase enzyme preparation of the invention are due to the high production of highly active phytase enzyme into the medium by the production strain Aspergillus niger 921, CCF 2951 and the more advantageous economy of the phytase enzyme production process compared to other similar published processes.

The invention is further illustrated by the following non-limiting examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

A high-production industrial strain of Aspergillus niger 921, CCF 2951 CZ patent 281581 was used. A conidia suspension from grown and sporulated culture on sloping sweet agar was an inoculum for submersive strain cultivation in 500 mL culture flasks. The cultivation of the strain in the first step was carried out on an 8 wt. % corn starch and 0.86 wt. sodium nitrate, limiting cell growth by phosphorus (Shieh and Ware 1968), on a shaker at 30 ° C. Laboratory bioreactors were inoculated with the grown mycelium. Strain cultivation in the second stage was carried out at 30 ° C, initial pH 5.3, stirring at 600 rpm. and aeration of 0.5 L / min. The laboratory bioreactors were inoculated with 75 L bioreactors.

Cultivation in 75 L bioreactors was carried out at pH 2.0-3.6, 30-35 ° C and pO _{2 of} 20-60% on production medium containing 1% w / w. lower starch content. During cultivation, the production of phytase enzyme, which is secreted into the medium along with non-specific acid phosphatases and α-amylase, was measured. After reaching phytase activity in the medium reaching 160 nkat / mL, the cooled bioreactor content was separated on a Sharpless separator.

The clear supernatant was dialyzed and ultrafiltered on a Romicon PM 50. During these processes, the reducing agent content of the supernatant was reduced to 2.0 g / L and the phytase activity increased on average 2.5 times.

-2GB 282772 B6

The concentrated and washed enzyme solution was lyophilized on a LZ 45 lyophilizer for 48 hours to dryness. The dry preparation containing in addition to phytase also non-specific acid phosphatase and α-amylase was diluted for feed purposes with wheat feed flour to phytase activity reaching 9 nkat / mg.

Example 2

The procedure was as in Example 1 except that the culture medium contained 0.86 wt. sodium nitrate sulphate or ammonium chloride in amounts corresponding to the concentration of nitrogen in sodium nitrate. During fermentation, the pH of the medium was regulated at a constant pH of 3.0 by automatic dosing of potassium, sodium or ammonium hydroxide solutions.

Example 3

The procedure was as in Example 1 or 2 except that the preparation of the inoculum for the operational bioreactors was three steps. The content of the 75 L bioreactor was after 48 h culture with an inoculum for cultivation in the 300 L bioreactor and afterwards. in a 1.5 m ³ bioreactor.

Example 4

The procedure was as in Example 1, 2 or 3 except that the cultivation in the process bioreactors was semi-continuous. A portion of the fermented fungal mycelium medium from the end of the fermentation was an inoculum for immediate subsequent fermentation in a parallel bioreactor.

Example 5

The procedure was as in the previous examples except that the cultivation in the process bioreactors was semi-sterile to non-sterile since the pH of the medium was in the range of 2.5-3.6 during cultivation.

Example 6

Proceed as in the previous examples, except that the separation of mycelium from the fermented medium is carried out in 2 steps. In the first step, the fermented medium is first deprived of most of the mycelium by sedimentation. In the second stage, the slightly turbid supernatant is separated on a centrifuge or filtered.

Example 7

Proceeding as in the previous examples, except that the dialysis and ultrafiltration of the fermented medium takes place on different types of devices and membranes other than ROMICON 50, which provide protein retention of 10-100 kDa molecules.

-3GB 282772 B6

Industrial applicability

The process according to the invention can be used for the fermentative industrial production of an enzyme preparation of phytase, which also contains non-specific acid phosphatases and α-amylase, useful in animal production, ecology, food industry, for the preparation of purified phytase, acid non-specific phosphatases and phosphorylated compounds.

PATENT CLAIMS

Claims (4)

Process for the fermentative industrial production of a phytase enzyme preparation by submersible cultivation using Aspergillus strain on a starch-containing medium with limitation of phosphorus growth, characterized in that the Aspergillus niger 921 CCF 2951 strain is cultivated at 25-37 ° C, pH 2.0- 6.0, especially 2.5-3.5, with aeration and stirring providing an oxygen concentration of 10-99% gas solubility coefficient, especially at 20-60%, followed by separation of the fungal mycelium, dialysis and ultrafiltration of the supernatant, and lyophilization of the concentrated enzyme solution . Method according to claim 1, characterized in that the culture medium contains 0.5-1% by weight. sodium nitrate, ammonium sulfate or ammonium chloride and during culture in the presence of ammonium sulfate or ammonium chloride, the pH of the medium is maintained at pH 2.5-3.5 by automatic dosing of potassium, sodium or ammonium hydroxide solutions. Method according to claim 1, characterized in that a medium containing 5-9 wt. % starch and, for production fermenters, a medium containing 4-8 wt. corn starch. The method of claim 1, wherein the strain is cultured semi-continuously under semi-sterile to non-sterile conditions.